Transformation-associated recombination (TAR) cloning is a method for isolating a large chromosomal region from a mammalian genome which relies on yeast Saccharomyces cerevisiae. In TAR cloning, total genomic DNA is extracted from a cell and co-transformed into yeast cells along with a vector carrying targeting sequences specific to a gene of interest. Upon co-transformation into yeast, homologous recombination occurs between the vector' hooks and targeted genomic sequences flanking the gene of interest to form a circular YAC (Yeast Artificial Chromosome). Chromosomal regions with sizes ranging to 250 kb have been isolated by TAR. Subsequent manipulation is required if sequences are required in a non-YAC vector, such as movement of sequence from a YAC to a BAC for downstream use.
Reference is made to Nat Commun. 2015 Sep. 1; 6:8101. doi: 10.1038/ncomms9101; “Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters”, Jiang W et al. The authors described a cloning method in which a target genome segment is excised from bacterial chromosomes in vitro by the RNA-guided Cas9 nuclease at two designated loci, and ligated to a cloning vector by Gibson assembly.
Engineered CRISPR/Cas systems have been used for precise modification of nucleic acid in various types of prokaryotic and eukaryotic cells, ranging from bacterial to animal and plant cells (eg, see Jiang W et al (2013)). Cutting of target nucleic acid has been proposed to generate recombinogenic, free ends in target nucleic acid. These ends promote homologous recombination with partner nucleic acid by homology-directed repair (HDR) mechanisms, eg, for insertion of partner sequence into the target. Current Cas-based techniques are, however, limited by the need to scour host cell genomes (or other target nucleic acids) to find PAM sites to match the desired Cas and then to select only those that are adjacent sufficiently unique protospacer sequences in the genome. Ideally, the protospacer/PAM combination should be unique to minimise the chances of off-target cutting, but this can be difficult to achieve in practice. This limits utility for gene therapy in human and other settings where accuracy is paramount. Furthermore, the need to use sites already in the genome reduces flexibility to freely target desired areas of the genome for homologous recombination.